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Journal of Animal Husbandry and Dairy Science
Volume 5, Issue 3, 2018, PP 32-38
ISSN 2637-5354
Journal of Animal Husbandry and Dairy ScienceV2 ●I3 ●2018 32
Pathomorphological and Immuno histochemical Findings of
Different Types of Cutaneous Papillomas in Bovines
P.J.Shruthi, K.Sujatha*, Ch.Srilatha and V.C. Rayulu
1
Department of Veterinary Pathology, College of Veterinary ScienceSri Venkateswara Veterinary
University, Tirupati – 517502 ( AP) 1 Department of Veterinary Parasitology, CVSc, Tirupati
*Corresponding Author: K.Sujatha, Department of Veterinary Pathology, College of Veterinary
Science Sri Venkateswara Veterinary University, Tirupati – 517502 (AP).Email:
INTRODUCTION
In cattle, papillomatosis is caused by bovine papilloma viruses (BPV), belonging to genus
Papillomavirus, family Parovaviridae. This
genus also includes viral agents causing papillomatosis in other animal species (goats,
dogs, rabbits, horses, and rodents) and men. The
virion has an icosahedral symmetry, size of 55 nm and contains a single two-stranded DNA
molecule 7-8 kb of length (Zarkov, 2003).
Twelve BPV serotypes are known – BPV 1-12,
causing diseases with various clinical manifestations. Bovine papillomatosis is caused
by BPV types from 1 to 10 (Vidya et.al. 2009),
but BPV-1 and BPV-2 are outlined as main agents of fibropapillomatosis in cattle due to
their affinity to epithelial tissue and skin
(Pangty, et.al. 2010 and Singh, et.al. 2009).
Under appropriate conditions, BPV-1 and BVP-2 could also cause fibroblastic papillomas in
horses (Nasir and Camoi, 2008), as well as
cutaneous sarcoid and non-regressing neoplasm in this species (Ragland and Spencer., 1968).
BPV infection results from the replication of the
virus in basal cells and subsequent formation of wart-like growths, most of which are benign and
do not proliferate infinitely. The localisation of
growths is different: abdominal and thoracic
wall, udder, vulva, head, neck etc. Young
animals < 2 years of age are most commonly affected, although papillomatosis is also
encountered in adult cattle. It is prevalent in
both genders and all breeds. Heifers are more
frequently affected than steers.
MATERIALS AND METHODS
Source
The materials for present study included the
samples suspected for neoplasia of bovine origin
submitted to the Department of Veterinary
Pathology, College of Veterinary Science, Tirupati by the Department of Veterinary
Surgery and Radiology and Veterinary
Dispensaries in and around Tirupati. Samples were also collected from animals necropsied in
the Department of Veterinary Pathology.
Particulars like breed, sex, location, shape,
colour and consistency of growths were recorded.
Histopathology
Representative tissue samples collected were fixed in 10 percent neutral buffered formalin
and processed routinely for histopathological
examination. Sections of 4-6 µ thickness were made and stained with Haematoxylin and Eosin.
ABSTRACT
Bovine Papillomatosis (BP) is a cutaneous viral disease of cattle caused by Bovine Papilloma Virus
(BPV).Out of 57 tumor samples, 12 were diagnosed as papillomas or warts (21.05%) of which ten were
collected from cattle and two from buffaloes. The animals had either generalized or solitary cutaneous
growths in different parts of the body. Grossly, the growths were solitary or multiple, pinkish to greyish-white in colour, 0.5 to 10 cm in diameter, sessile or pedunculated and hard in consistency.
Histopathologically, squamous papilloma, viral papilloma (papillomatosis), fibropapilloma and
angiokeratoticacanthomatous papilloma were noticed. Immuno histo chemically, PCNA expression in
papilloma was observed predominantly in the cells of basal and parabasal layers of epidermis. The PCNA
antibodies were very useful marker to detection of cellular proliferation in the bovine cutaneous papillomas
Pathomorphological and immunohistochemical findings of different types of cutaneous papillomas in
bovines
33 Journal of Animal Husbandry and Dairy ScienceV2 ●I3 ●2018
Special staining technique with Masson’s
Trichome, was employed (Luna, 1968 and Bancroft and Cook, 1994).
IMMUNOHISTOCHEMISTRY
Immunohistochemistry was performed using
Universal Dakocytomation LSAB® kit to
evaluate the expression of proliferative markers
namely PCNA.
Procedure
Sections of 5-6 µ thickness were mounted on to
APES (Amino Propyl EthoxySialine) coated slides and incubated overnight at 37ºC.Sections
were deparaffinized and rehydrated.Sections
were kept in citrate buffer (10mM, pH 6) and subjected to microwave treatment for 5 cycles
each for 5 minutes at 750 watts to retrieve the
antigenic sites.Sections were allowed to cool
down to the room temperature.Sections were rinsed in TBS (Tris Buffer Saline).One drop of
hydrogen peroxide was kept over the section for
15 minutes to block unwanted antigenic sites and to quench endogenous peroxide activity.
Sections were rinsed with TBS. Primary
antibody was added onto the sections and
incubated for 30 minutes. Sections were rinsed with TBS. Sections were incubated with
secondary link antibody for 30 minutes.
Sections were rinsed with TBS. Sections were incubated with tertiary Streptavidin peroxidase
for 30 minutes. Sections were rinsed with
TBS.DAB (Diaminobenzidine) was used as chromogen and the sections were incubated for
10 minutes. Sections were rinsed with distilled
water. Sectionswere counter stained with Harris
haematoxylin and mounted in DPX.
RESULTS
Out of 57 tumor samples, 12 were diagnosed as papillomas or warts (21.05%) of which ten were
collected from cattle (two from Holstein
Friesian, one from Jersey, three from Hallikar
and four from non-descript cattle) and the remaining two from buffaloes. Out of 11 cases
of papilloma, there were nine cases of squamous
papilloma and viral papilloma (papillomatosis), two cases of fibropapilloma (in a Jersey bull and
a Hallikar bullock) and one case of
angiokeratoticacanthomatous papilloma (in a non-descript cow). The incidence of papilloma
was equal in male and female animals.
The animals had either generalized or solitary
cutaneous growths in different parts of the body like head, neck, shoulder, limbs, udder and teats,
scrotum and inner aspect of thigh.
Macroscopically the growths were solitary or multiple, pinkish to greyish-white in colour, 0.5
to 10 cm in diameter, sessile or pedunculated
and hard in consistency. The surface was either smooth or rough and horny (Fig. 1 to 6).
Fig1. Squamous papilloma: HF cow: Multiple,
grayish, roughy and horny growths around the eye.
Fig2. Squamous papilloma: ND cow: Multiple gray
to pink coloured warts with rough and horny surface
on head, neck and shoulder region.
Fig3. Squamous papilloma: Cow: Small, solitary
growths with smooth surface on udder and teats.
Fig4. Squamous papilloma: Bullock: Note multiple
pinkish pedunculated growths near scrotum.
Pathomorphological and immunohistochemical findings of different types of cutaneous papillomas in
bovines
Journal of Animal Husbandry and Dairy ScienceV2 ●I3 ●2018 34
Fig5. Fibropapilloma: Note multiple warts which are
round, sessile, dome shaped with a rough and even
surface around the eye.
Fig6. Angiokeratotic papilloma: Jersey cow:
Multiple, grayish pink, rough and horny growths on
the ear and neck.
Histopathological examination revealed that the epidermis was thickened and hyperplastic with
elongated, finger like outward projections in
squamous papilloma (Fig. 7) and irregular, elongated, inward rete pegs in papillomatosis
(viral papilloma) (Fig. 8) with varying degrees
of parakeratosis, hyperkeratosis, acanthosis (Fig. 9) and a core of dermal fibrous connective
tissue. The dermal papillae were elongated with
irregular rete ridge formation deep into the
dermis with lateral interconnections forming islands of dermal connective tissue surrounded
by hyperplastic epidermal cells. The
keratinocytes within stratum spinosum showed clear hydropic degeneration and a clear
perinuclear halo with pyknotic nucleus
(koilocytes). The granular cell layer with
prominent basophilic keratohyaline granules was observed and the basal cell layer was
hyperplastic. Occasionally melanin granules
were found freely within the subepidermal and dermal melanophages. In fibropapilloma,
extensive proliferation of fibroblasts and
epidermal keratinocytes was noticed (Fig. 10 and 11). The dermal layer revealed proliferating
cells which were large, plumb fibroblasts,
arranged in haphazard whorls and fascicles. In
angiokeratotic papilloma, there were discrete
groups of variably sized, dilated, thin walled and well differentiated vascular channels which
were filled with blood and lined by a single
layer of flattened endothelial cells (Fig. 12).
Fig7. Squamous papilloma: Finger like outward
projections of epidermis with parakeratosis, acanthosisand a core of fibrous connective tissue.
Note koilocytes with a perinuclear halo and a
pyknotic nucleus. H&E: x 70.
Fig8. Papillomatosis: Section showing irregular,
elongated inward growths of rete pegs deep
into the dermis with lateral interconnections. H&E: x
70.
Fig9. Papilloma: Note finger like projection from
epidermis with increased thickness, parakeratosis
and hydropic degeneration of keratinocytes. H&E: x 280
Pathomorphological and immunohistochemical findings of different types of cutaneous papillomas in
bovines
35 Journal of Animal Husbandry and Dairy ScienceV2 ●I3 ●2018
Fig10. Fibropapilloma: Note the finger like
projection from epidermis with parakeratosis and
extensive proliferation of fibroblasts in the dermis.
H&E: x 70.
Fig11. Fibropapilloma: Note the characteristic
thickening of epidermis with parakeratosis (red) and fibrous tissue proliferation (blue) in the dermis.
Masson’s Trichome stain: x 70.
Fig12. Angiokeratotic papilloma: Finger like
projections of epidermis with groups of dilated, thin
walled vascular channels filled with blood. H&E: x
70.
Fig13. Papilloma: IHC: Section showing positive
immunostaining in the nuclei of basal, parabasal
cells and connective tissue. PCNA: x 70.
Fig14. Papilloma: IHC: Section showing positive
immunostaining in the nuclei of basal, parabasal
cells and connective tissue. PCNA: x 280.
IMMUNOHISTOCHEMISTRY
Positive expression of PCNA was observed in
90% (9/10) of tumors assessed and the protein expression was restricted to the nucleus. In
papillomas, PCNA expression was detected
predominantly in the basal and parabasal layer
in two cases and in one case positive immunostaining was seen in the nuclei of
epidermal cells and connective tissue (Fig. 13
and 14).
DISCUSSION
In the present study, papillomas were observed
in 21.05% of the cases. Sivaseelan et al. (2009) reported the incidence of papillomas in domestic
animals as 4.34%. There were nine cases of
squamous papilloma and papillomatosis (viral papilloma), two cases of fibropapilloma and one
case of angiokeratoticacanthomatous papilloma.
Similar rate incidence was observed in both male and female animals in the current study.
However, Ozsoy et al. (2011) observed a
slightly high incidence in males (54.9%) than in
the female (45.1%) cattle. This disease might be due to the spread of BPV via direct contact,
contaminated feed and equipment, castration
and injections. Inheritance, nutritional and hormonal disorders, sunlight and suppressed
immune system might have also played a role in
the pathogenesis as reported earlier by Campo et al. (1994), Otter and Leonard (2003) and Frietas
et al. (2011).
The papillomas or warts were either generalized
or solitary and located in different parts of the body like head, neck, shoulder, limbs, udder and
teats, scrotum and inner aspect of thigh. (Blood
and Radostits, 1989; Jones et al., 1997; Veena, 2001; Goldschmidt and Hendrick, 2002; Veena
and Ravi Kumar, 2002; Hatama, 2011; Tan et
al., 2012). The surface was either smooth or
Pathomorphological and immunohistochemical findings of different types of cutaneous papillomas in
bovines
Journal of Animal Husbandry and Dairy ScienceV2 ●I3 ●2018 36
rough and horny. Similar findings were reported
by Wadhwa et al. (1995), Veena (2001), Wangikar et al. (2001), Gulbahar et al. (2003),
Sood et al. (2007), Debasis Jana and Mousumi
Jana (2009), Pawan Kumar et al. (2010), Ozsoy et al. (2011), Vidya Singh et al. (2010), Tan et
al. (2012) and Tozato et al. (2013).
Histopathological examination of papillomas revealed that the epidermis was thickened and
hyperplastic with elongated, finger like outward
projections in squamous papilloma and
irregular, elongated, inward rete pegs in papillomatosis with varying degrees of
parakeratosis, hyperkeratosis, acanthosis and a
core of dermal fibrous connective tissue. A clear hydropic degeneration or a perinuclear halo and
pyknotic nucleus was observed in keratinocytes
(koilocytes). These lesions were in accordance with the observations of Wangikar et al. (2001),
Vidya Singh et al. (2010), Ozsoy et al. (2011),
Tan et al. (2012) and Tozato et al. (2013). There
was an extensive proliferation of fibroblasts and epidermal keratinocytes in fibropapillomas.
Correspondingly, Theilen et al. (1985),
Abdouslam et al. (1997), Vikas (2010), Bam et al. (2012), Tan et al. (2012) and Pawan Kumar
et al. (2013) observed similar findings in
fibropapilloma. Discrete groups of variably
sized, dilated, thin walled and well differentiated vascular channels, filled with blood and lined by
a single layer of flattened endothelial cells were
noticed in angiokeratotic papilloma. Similar changes were observed by Gulbahar et al.
(2003), Bharath (2007) and Sood et al. (2007).
In the present study, PCNA expression in papilloma was observed predominantly in the
cells of basal and parabasal layers of epidermis
and in one case the expression was seen in both
epidermal cells and connective tissue. These results were in conformity with the findings of
Pawan Kumar et al. (2013).
In conclusion, the present study revealed that incidence of different types of cutaneous
papillomas in both cattle and buffaloes. There
was no sex predilection was recorded and cases were reported in cross bred and non descriptive
cattle. The papillomas were noticed in different
parts of the body. Histopathologically, squamous
papilloma, viral papilloma (papillomatosis), fibropapilloma and
angiokeratoticacanthomatous papilloma were
noticed. Immunohistochemically, PCNA expression in papilloma was observed
predominantly in the cells of basal and parabasal
layers of epidermis. The PCNA antibodies were
very useful marker to detection of cellular proliferation in the bovine cutaneous
papillomas.
ACKNOWLEDGEMENTS
The authors are very much thankful to Sri
Venkateswara Veterinary University, Tirupati for
providing the facilities to carry out the present MVSc research work.
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Citation: P.J.Shruthi, K.Sujatha, Ch.Srilatha and V.C. Rayulu“ Pathomorphological and
Immunohistochemical Findings of Different Types of Cutaneous Papillomas in Bovines” Journal of
Research in Animal Husbandry and Dairy Science, 2(3), pp 32-38.
Copyright: © 2018 K.Sujatha. This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.